Fluorescence Imaging of Intracellular Telomerase Activity Using Enzyme-Free Signal Amplification

Abstract

A novel enzyme-free signal amplification-based assay for highly sensitive in situ fluorescence imaging and detection of intracellular telomerase activity was developed by using a gold nanoflare probe-triggered mimic-hybridization chain reaction (mimic-HCR) coupled with a graphene oxide (GO) surface-anchored fluorescence signal readout pathway. The nanoflare probe consists of gold nanoparticles (AuNPs) functionalized with a dense shell of nucleic acid sequences by Au-S bond formation. The nucleic acid sequence is composed of three segments: a long thiol-labeled sequence (HS-DNA) and two short sequences (a telomerase primer sequence, "Primer-DNA", and an FAM-terminated reporter sequence, "Flare-DNA"), both of which are complementary to HS-DNA. The mimic-HCR system is formed by two FAM-modified hairpin sequences that are adsorbed on GO. Upon endocytosis of the AuNP/GO combinatorial probe, the Primer-DNA can be extended by intracellular telomerase at its 3' end to produce the telomeric repeated sequence, which leads to inner chain substitution and not only releases the Flare-DNA to turn on the fluorescence of FAM but also initiates the subsequent signal amplification and enrichment for the mimic-HCR system anchored on GO. The proposed approach can sensitively detect telomerase activity in living cells, distinguish normal cells from cancer cells, and monitor the change in telomerase activity in response to a telomerase inhibitor.

Biomedical applications of nanoflares: Targeted intracellular fluorescence probes

“…AuNPs-based nanoflares with acceptor-donor flare beacon 97 with permission. Schematic representation of AuNP-GO hybrid-based nanoflares 99 with permission. Telomerase activity detection using ratiometric nanoflares 100 with permission.…”

“…A schematic illustration of AuNPs-GO hybrid-based nanoflares is shown in Figure 7. 99 In another study, He and co-workers, developed a ratiometric fluorescent-based biosensor for detection of cancer cells based on differences in telomerase expression levels. They prepared modified AuNP-based nanoflares with oligonucleotides to detect telomerase mRNA in cancer (HeLa, HepG2, A549, and 293 T) and normal (QSG) cells.…”

Expression of microRNA-155 and human telomerase reverse transcriptase in patients with bladder cancer

“…Telomerase activity is inhibited in normal human cells and telomeres are progressively shortened after each replication cycle, resulting in cell senescence and death [5]. However, telomerase is reactivated and overexpressed in over 85% of malignant cancer cells, allowing them to survive and proliferate uncontrollably [6]. Therefore, telomerase has been considered as a potential biomarker and as a promising therapeutic target for cancer [7].…”

Cascaded Electrochemiluminescence Signal Amplifier for the Detection of Telomerase Activity from Tumor Cells and Tissues

“…As theoretically infinitely proliferous organisms, tumor cells can readily proliferate 14 - 16 , and this is usually accompanied by systematic and complicated changes in the intracellular environment 17 , 18 . In particular, telomerase is overexpressed in most cancer cells 19 - 21 , while it is absent from or present in very low levels in normal human somatic cells 22 - 24 . These studies indicate that telomerase plays a crucial role in tumorigenesis, and it has been employed as a potential biomarker for prognosis, tumorigenesis and metastasis 25 - 27 .…”